Bio-Rad ProteinChip Antibody Capture Kit User Manual

Download

ProteinChip®Antibody Capture Kit

Instruction Manual

Catalog #K100-0005

For technical support, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723).

Introduction

Traditional immunoassay-based protein detection methods are often difficult to develop and are complicated by antibodies cross-reacting and binding nonspecifically to a variety of proteins and peptides. The ProteinChip antibody (Ab) capture kit enables unambiguous species-specific protein identification and epitope discovery experiments on one platform, and eliminates the need to develop a sandwich assay. Using the components provided in the kit, antibodies are bound to a ProteinChip array to detect antigen(s) of interest from crude samples derived from in vivo or in vitro experimental models. Once an antibody-antigen complex has been formed on the ProteinChip array, researchers can use the ProteinChip array for two unique purposes: 1) to identify proteins bound to the antibody on the basis of molecular weight using the ProteinChip SELDI reader, or 2) to conduct epitope discovery experiments by proteolytically cleaving proteins captured on ProteinChip arrays (on-chip proteolysis) and then using SELDI to highlight the peptide(s) associated with the epitope, streamlining the process of sequencing the protein binding site via tandem mass spectrometry (MS/MS).

Fig. 1. Principles of the ProteinChip antibody capture kit. Protein G is covalently bound to the ProteinChip PG20 array surface. An optional crosslinking step can be performed to crosslink IgG antibodies to protein G.

The key component of this kit is the ProteinChip PG20 array (ProteinChip PS20 array precoupled with recombinant protein G (Pierce). Any antibody capable of binding protein G can be used with the kit (see appendix for details). The kit also contains positive (TNF-α antigen-antibody pair) and negative (bovine IgG) controls and a crosslinking reagent to bind antibodies covalently to ProteinChip PG20 arrays.

Materials

Materials Included

TNF-α antibody (0.2 mg/ml anti-human TNF-α goat polyclonal antibody in phosphate buffered saline (PBS), 20 µl

TNF-α antigen* (1.0 µM recombinant human TNF-α antigen in 0.1% bovine serum albumin (BSA)/PBS), 10 µl

Bovine IgG (lyophilized), 750 µg

Wash buffer (0.5% Triton X-100 in PBS); 2 bottles, 120 ml each

PBS; 2 bottles, 120 ml each

1x PBS; 1 bottle, 4 ml**

ProteinChip PG20 arrays, 6

Bis(sulfosuccinimidyl) suberate (BS3) crosslinking reagent (0.5 mg/vial), 2

Deactivation buffer (0.5 M ethanolamine in PBS, pH 8.0), 4 ml

ProteinChip sinapinic acid (SPA) energy absorbing molecules (EAMs), 5 mg

Instruction manual

* Contains equimolar amounts of TNF with and without N-terminal methionine, yielding a double peak.

** The 4 ml PBS should be used to dilute the bovine IgG, antibody, and antigen, reconstitute the

crosslinking reagent, and deposit to the spots of the ProteinChip array when needed.

aterials Needed but Not Included

15 ml conical tubes

Rocking platform

Humidity chamber*

Lab timer

Ice bucket and ice

Microcentrifuge tubes

Calibrated pipets**

99+% trifluoroacetic acid (TFA)

Optional Materials

ProteinChip cassette-compatible bioprocessor (catalog #C50-30011)

MicroMix 5 plate and tube shaker*** (Diagnostic Products Corporation)

Sonicating water bath

Shipping and Storage

Table 1. Shipping and storage conditions for ProteinChip antibody capture kit components.

Item Shipping Storage

Anti-human TNF-α Dry ice –20°C (stable 6 months) antibody 4°C (stable 2 weeks)

Recombinant human Dry ice –20°C (stable 6 months)

α antigen 4°C (stable 2 weeks)

TNF-

Bovine IgG Dry ice –20°C

Crosslinking reagent Dry ice –20°C

ProteinChip PG20 arrays Ambient with desiccant 4°C with desiccant

Wash buffer Ambient 4°C

PBS Ambient 4°C

Deactivation buffer Ambient 4°C

HEPES buffer Ambient 4°C

ProteinChip SPA EAMs Ambient Ambient or 4°C

* See the ProteinChip SELDI

humidity chamber if one is not r

System Applications Guide for detailed instructions on how to create a

eadily available.

** Calibrated pipets will yield more reproducible results.

*** When using a ProteinChip bioprocessor, a MicroMix 5 is recommended.

Protocol Flowchart

Step 1: Coupling Antibody to the ProteinChip Array

1. Thaw the TNF-α antibody and TNF-α antigen on ice.

2. Reconstitute 750 µg of the bovine IgG provided in the kit with 100 µl PBS to

obtain a final concentration of 7.5 mg/ml.

3. Add 4 µl reconstituted control bovine IgG to 146 µl of PBS to obtain a final concentration of 0.2 mg/ml.

4. Place a ProteinChip PG20 array on a clean, flat surface.

5. Add 2 µl of the diluted bovine IgG to spot A on the array.

6. Add 2 µl of the TNF-α antibody to spot B on the array.

7. Add 2 µl 0.2 mg/ml antibody of interest to the remaining spots on the array.

8. Immediately transfer the array to a humidity chamber and incubate for 1 hour at

room temperature or overnight at 4°C. If larger sample volumes are being used with a ProteinChip bioprocessor, incubate on a rocking platform.

9. Remove the bovine IgG and the TNF-α antibody from the array using a pipet to prevent cross-contamination during the following wash steps. Do not touch the surface of the array with the pipet tip.

10. Place the entire array into a 15 ml conical tube containing 8 ml wash buffer and agitate on a rocking platform for 10 minutes at room temperature.

11. Pour off the wash buffer and add 8 ml PBS to the tube and agitate on a rocking platform for 5 minutes at room temperature.

12. Pour off the PBS and repeat step 1.11 once for a total of two washes.

13. After the PBS washes, gently blot the excess buffer from the array surface

using a lint-free lab wipe without touching the active spots on the array.

Step 2: Crosslinking Antibody to Protein G (Optional)

1. Reconstitute the crosslinking reagent as follows: Warm the crosslinking reagent to room temperature, dissolve 0.5 mg (one vial) of crosslinking reagent by adding 1 ml of PBS into the vial, and vigorously vortex the vial.

2. After removing the buffer from the array (step 1.13), air-dry the array for approximately 5 minutes. Do not allow the spots to dry completely. If the spots dry, add 1 µl PBS to each spot on the array.

3. Add 1 µl of crosslinking reagent to each spot on the array.

4. Immediately transfer the array to a humidity chamber and incubate for

30 minutes at room temperature.

5. Add 1 µl deactivation buffer to each spot, transfer the array to the humidity chamber, and incubate with agitation for 15

6. Blot the excess buf

fer from the surface of the array using a lint-free lab wipe

without touching the active spots on the ar

minutes at room temperature.

ray

7. Place the entire array in a 15 ml conical tube containing 8 ml of wash buffer. Agitate on a rocking platform for 10 minutes. Pour off the wash buffer.

8. Pour off the wash buffer and add 8 ml of PBS to the conical tube. Agitate vigorously on a shaker for 5 minutes.

9. Repeat step 2.8 for a total of two PBS washes.

Step 3: Capturing Antigens on the ProteinChip PG20 Array

1. After completing the PBS washes, pour off the buffer. Using a lint-free lab wipe, gently blot away any excess buffer from the surface of the array without touching the active spots on the array.

2. Dilute 1 µl of TNF-α antigen into 19 µl of PBS, and vortex. The final concentration will be 50 fmol/µl.

3. Add 2 µl (100 fmol) of the diluted TNF-α antigen to spots A and B on the array.

4. Add 2 µl of sample to the remaining spots on the array (C–H), and transfer the

array to a humidity chamber.

5. Incubate the array for 1 hour at room temperature, or incubate overnight at 4°C.

6. Place the array in a 15 ml conical tube containing 8 ml of wash buffer. Agitate on a rocking platform for 10 minutes. Pour off the wash buffer.

7. Add 8 ml of PBS to the conical tube. Agitate on a rocking platform for 5 minutes at room temperature.

8. Pour off the PBS, add 8 ml fresh PBS, and agitate for 5 minutes.

9. Make 500 ml of a 1 mM HEPES solution by adding 500 µl of 1 M HEPES to

500 ml deionized water.

10. Pour off the PBS and rinse the array two times with 1 mM HEPES.

11. Remove the array from the conical tube, flick off the HEPES, and allow to

air-dry for approximately 10 minutes.

Step 4: EAM Preparation and Addition

1. Add 100 µl 99.8% acetonitrile and 100 µl 1.0% TFA into the vial containing ProteinChip SPA EAM powder to obtain a final concentration of 25 mg/ml.

2. Vortex the vial for 5 minutes to dissolve the EAM powder. Alternatively, incubate the vial for 5 minutes in a sonicating water bath.

3. Add 0.5 µl of this saturated EAM solution to each spot on the array and air-dry.

4. Repeat step 4.3 and air-dry the array completely (approximately 10 minutes)

before reading in a Pr

oteinChip SELDI r

eader

Step 5: Reading the ProteinChip PG20 Array in a ProteinChip SELDI Reader

After the EAM has dried on the array, read the array in a ProteinChip SELDI reader. The spot protocol used to read the array can be created once and saved. The data collection parameters of the spot protocol are a function of molecular mass and need to be adjusted for each antigen. See the operation manual for ProteinChip data manager software for detailed instructions on how to create an optimized spot protocol. When optimizing the protocol for the detection of TNF-

α antigen (molecular mass 17.5 kD), set the Mass Range limits from 0 to 200,000 Da, and the Focus Mass to 17,500 Da.

Ordering Information

Catalog # Description

K10-00005

C55-30058 C50-30011

C30-00002

ProteinChip Antibody Capture Kit, includes antibody, antigen, reagents, controls, 6 ProteinChip PG20 arrays, ProteinChip SPA energy absorbing molecules (EAMs), instructions

ProteinChip PG20 Array, A–H format ProteinChip Cassette-Compatible Bioprocessor, includes ProteinChip array forceps,

cassette hold-down frame, 12 blank ProteinChip arrays ProteinChip SPA Energy Absorbing Molecules (EAMs), 5 mg/vial, 20

Appendix A

Immunoglobulin Binding Affinity to Protein G

The information in Table 2 is provided as a guideline and does not guarantee that all antibodies will behave identically to the information provided in the table.

able 2: Binding affinity of various immunoglobulins to protein G.

pecies Immunoglobulin Binding Affinity**

Human Total IgG S

I IgG IgG IgG

S S S S

Mouse Total IgG S

IgG IgG IgG IgG

M S S S

Rat Total IgG M

IgG IgG IgG IgG

M S W S

Bovine Total IgG S

IgG1 S IgG2 S

Cat Total IgG W

Dog Total IgG W

Goat Total IgG S

IgG IgG

S S

Guinea pig Total IgG W

Horse Total IgG S

IgG

(ab)

IgG

(c)

IgG(T) S

NB MB

Pig Total IgG W

Rabbit Total IgG S

Sheep Total IgG S

IgG IgG

S S

* Information provided by Pierce. ** W, weak binding; M, medium binding; S, strong binding; NB, no binding.

Spectra of TNF-α Antigen Capture

0.2

0.1

-0.1

0.6

0.4

0.2

-0.2

0.4

0.2

1.0

0.5

1.0

Intensity

0.5

7.5

5.0

2.5

1,400

Fig. 2. Spectra illustrating the ef

of TNF-

α is 17.5

kD. TNF-

α quantities range fr

1,600

m/z, kD

fect of increasing TNF-a quantity on intensity.

om 130 amol to 532 fmol. The double peak in these

1,800

20,000

The molecular mass

spectra represents equimolar amounts of TNF-a with and without N-terminus methionine.

ovalently Crosslinking IgG Antibodies to Protein G

Antibodies captured by protein G on ProteinChip PG20 arrays are not covalently bound to the surface of the array. When these arrays are analyzed in a ProteinChip SELDI reader, peaks may be seen in the resulting spectra corresponding to intact IgG (MH+ ~148 kD, M2H+ ~75 kD) or its fragments (~75 kD, ~48 kD, ~22 kD), especially if a high laser energy is used. These peaks may interfere with the detection or quantitation of an antigen of similar molecular weight. When the antibody is crosslinked to protein G on the array, it becomes covalently attached to the array surface and will not be desorbed from the array surface during the reading process.

The results of an antibody capture assay depicting crosslinked and noncrosslinked antibody are shown in Figure 3. Without crosslinking, an IgG peak (molecular mass 148 kD) is often seen. When the IgG is crosslinked to the ProteinChip PG20 array, the IgG peak is not observed. Crosslinking does not affect the peak intensity of TNF-α antigen.

.75

.50

.25

15,000

Intensity

.075

.050

.025

120,000

Not Crosslinked

17,500

140,000 160,000

20,000

15,000 17,500

120,000

m/z, kD

Crosslinked

20,000

140,000 160,000

Fig. 3. Effect of crosslinking on TNF-α antigen and TNF-α antibody peak profiles. A, peak corresponding to the 17.5 kD protein, TNFPG20 array; B, peak corresponding to the 145 kD TNFapplied to a ProteinChip PG20 array; C, peak corresponding to the 17.5 kD protein, TNFwhen 100 fmol TNFthe array; D, no peak is seen corresponding to the 145 kD TNFis added to a ProteinChip PG20 array.

formance Specifications

Per

α is applied to a ProteinChip PG20 array and the anti-TNF-α antibody is crosslinked to

α, is seen when 100 fmol TNF-α is applied to a ProteinChip

α IgG antibody is seen when 2.7 pmol of antibody is

α, is seen

α IgG antibody when 2.7 pmol of antibody

Using the reagents supplied with the ProteinChip antibody capture kit, the user should be able to detect the TNFhuman TNF-

α antigen in 0.1% BSA/PBS) down to a level of 1 fmol at a signal-to-

noise ratio of at least 3. For the TNF-

α antigen positive control (1.0 µM recombinant

α control, the quantitation range will generally

be from 10–500 fmol.

Different antigens behave significantly differently in the antibody capture and SELDI processes, so these values cannot be applied to any other antigen-antibody system. The lower limit of detection and dynamic range for each antigen needs to be determined for each system.

MicroMix is a trademark of Diagnostic Products Corporation. Triton is a trademark of Union Carbide.

Life Science Group

06-0733 0307 Sig 1106

10008252 US/EG Rev D

Bio-Rad Laboratories, Inc.

Web site www.bio-rad.com USA 800 4BIORAD Australia 61 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 3237 9400 Canada 905 712 2771 China 86 21 6426 0808 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000

The SELDI process is covered by US patents 5,719,060, 5,894,063, 6,020,208, 6,027,942, 6,124,137, 6,225,047, 6,528,320, 6,579,719, and 6,734,022. Additional US and foreign patents are pending.

Bio-Rad ProteinChip Antibody Capture Kit User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual